Guan Xin, Madabushi Amrita, Chang Dau-Yin, Fitzgerald Megan E, Shi Gouli, Drohat Alexander C, Lu A-Lien
Department of Biochemistry and Molecular Biology and Greenebaum Cancer Center, School of Medicine, University of Maryland, Baltimore, MD 21201, USA.
Nucleic Acids Res. 2007;35(18):6207-18. doi: 10.1093/nar/gkm678. Epub 2007 Sep 12.
Human (h) DNA repair enzyme thymine DNA glycosylase (hTDG) is a key DNA glycosylase in the base excision repair (BER) pathway that repairs deaminated cytosines and 5-methyl-cytosines. The cell cycle checkpoint protein Rad9-Rad1-Hus1 (the 9-1-1 complex) is the surveillance machinery involved in the preservation of genome stability. In this study, we show that hTDG interacts with hRad9, hRad1 and hHus1 as individual proteins and as a complex. The hHus1 interacting domain is mapped to residues 67-110 of hTDG, and Val74 of hTDG plays an important role in the TDG-Hus1 interaction. In contrast to the core domain of hTDG (residues 110-308), hTDG(67-308) removes U and T from U/G and T/G mispairs, respectively, with similar rates as native hTDG. Human TDG activity is significantly stimulated by hHus1, hRad1, hRad9 separately, and by the 9-1-1 complex. Interestingly, the interaction between hRad9 and hTDG, as detected by co-immunoprecipitation (Co-IP), is enhanced following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. A significant fraction of the hTDG nuclear foci co-localize with hRad9 foci in cells treated with methylating agents. Thus, the 9-1-1 complex at the lesion sites serves as both a damage sensor to activate checkpoint control and a component of the BER.
人类（h）DNA修复酶胸腺嘧啶DNA糖基化酶（hTDG）是碱基切除修复（BER）途径中的关键DNA糖基化酶，负责修复脱氨基胞嘧啶和5-甲基胞嘧啶。细胞周期检查点蛋白Rad9-Rad1-Hus1（9-1-1复合体）是参与维持基因组稳定性的监测机制。在本研究中，我们表明hTDG与hRad9、hRad1和hHus1分别作为单个蛋白以及作为复合体相互作用。hHus1相互作用结构域定位于hTDG的67-110位残基，hTDG的Val74在TDG-Hus1相互作用中起重要作用。与hTDG的核心结构域（110-308位残基）不同，hTDG(67-308)分别从U/G和T/G错配中去除U和T的速率与天然hTDG相似。hHus1、hRad1、hRad9单独以及9-1-1复合体均可显著刺激人类TDG活性。有趣的是，经N-甲基-N'-硝基-N-亚硝基胍（MNNG）处理后，通过免疫共沉淀（Co-IP）检测到的hRad9与hTDG之间的相互作用增强。在用甲基化剂处理的细胞中，很大一部分hTDG核灶与hRad9灶共定位。因此，损伤位点的9-1-1复合体既是激活检查点控制的损伤传感器，也是BER的一个组成部分。
